Regulation of intracellular protein trafficking is one of the central issues in cell biology. Extracellular proteins are delivered to cells via endocytosis, in which the macromolecules are sorted to their ultimate intracellular destination, often the lysosome for proteolysis. Under stress conditions, this vacuolar endosomal/lysosomal pathway is responsible for accelerated degradation of intracellular proteins via autophagic sequestration, whereas under basal conditions the major proteolytic fate of intracellular proteins is via the cytoplasmic ubiquitin system. during the past grant period, using temperature sensitive mutants of the ubiquitin pathway (defective in E1, the ubiquitin activating enzyme) we demonstrated a linkage between the endosomal-lysosomal system and the ubiquitin proteolytic system. Herein the initial and rate-limiting reaction of the ubiquitin pathway, E1, plays a pivotal role in this linkage. Thus, our long term goals are to elucidate the molecular mechanisms responsible for the cell biology of intracellular protein sorting and degradation. Our most recent studies show that E1 is a nuclear as well as cytoplasmic protein, exists as multiple species and its nuclear localization appears to vary in cell cycle. This raises questions focused on the role of E1 and the ubiquitin system in the nucleus. Furthermore, we have now shown that a major ubiquitinated nuclear protein, histone H2A, is recognized by essential and previously unknown proteins for ubiquitin conjugation/degradation. Thus, the specific aims f the present proposal are to elucidate (1) the structure and function of E1 and its role in the cell nucleus and cell cycle using cell and molecular approaches and (2) purification and determination of the mechanism(s) of action of the novel proteins responsible for ubiquitin conjugation/degradation of histone H2A using biochemical, cell and molecular biological approaches. These studies taken together will advance our understanding of the cellular biology of intracellular protein degradation.